Inhibition of cell invasion and migration by targeting matrix metalloproteinase-9 expression via sirtuin 6 silencing in human breast cancer cells

Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on protein kinase C activator- and cytokine-mediated cancer cell invasion and migration in MCF-7 and MDA-MB-231 cells and the association between SIRT6 and matrix metalloproteinase-9 (MMP-9) expression. To assess MMP-9 and SIRT6 expression in patients, protein levels in BC tissues were analyzed. MCF-7 and MDA-MB-231 cell viability was analyzed using MTT assays. SIRT6 was silenced in both cell lines and protein secretion, expression, and mRNA levels were analyzed. Transcription factor DNA activity was investigated using luciferase assays. Matrigel invasion assays were used to assess the effects of SIRT6 in both cell lines. SIRT6 and MMP-9 expression in cancer tissues was significantly higher than in paired normal breast tissues. 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) increased MMP-9 expression and cell invasion and migration, but SIRT6 knockdown abolished these effects. SIRT6 overexpression additively increased TPA- and TNF-α-induced MMP-9 expression. SIRT6 knockdown suppressed the mitogen-activated protein kinase (MAPK) signaling pathway and thus TPA- and TNF-α-induced MMP-9 expression. SIRT6 silencing suppressed TPA- and TNF-α-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) expressions in both cell lines, and treatment with MAPK, NF-κB, and AP-1 inhibitors reduced MMP-9 expression. The anti-invasive effects of SIRT6 in BC cells might be mediated by suppression of MAPK phosphorylation and reduction in NF-κB and AP-1 DNA activities, leading to MMP-9 downregulation, suggesting that SIRT6 modulation has the potential to target BC metastasis.

Transfection with small interfering RNA (siRNA). Duplexes of small interfering RNA (siRNA) targeting human SIRT6 mRNA (target sequences are as follows: si-SIRT6: CAG CUU AAA CAG GAG UGA A for sense and UUC ACU CCU GUU UAA GCU G for antisense). SIRT6-specific siRNA and negative control siRNA were obtained from BIONEER (Daejeon, Korea), Opti-MEM medium was obtained from Gibco, and Lipofectamine RNAimax was purchased from Invitrogen (Carlsbad, CA, USA). The cells were transfected according to the manufacturer's instructions (Invitrogen). Cells were harvested and re-suspended in Opti-MEM medium with Lipofectamine RNAimax, mixed with 50 pmol siRNA, and transfected for 12 h. SIRT6 knockdown was confirmed by western blotting and real-time polymerase chain reaction (RT-qPCR) assays.
Virus infection. Cells were placed in culture plates and infected with adenoviruses for 24 h in DMEM. The concentration of adeno-SIRT6 virus (multiplicity of infection, 300-500) used (as determined in a preliminary study) was equal to that of adeno-LacZ virus, which allowed infection of each cell line without toxic effects in a pre-experiment. Adenovirus expressing SIRT6 (provided by Professor Park Byeong-hyeon, Jeonbuk National University, Jeonju, South Korea), Production of AdSirt6 was described to in Park's paper 17 . Isolation of nuclear and cytoplasmic extracts. Transfected cells were treated with TPA or TNF-α for 3 h, washed with PBS, and pelleted by centrifugation. Nuclear and cytoplasmic extracts were prepared using NE-Zymography assay. Conditioned media were collected, mixed with sample buffer (non-reducing loading buffer), and separated by PAGE with gelatin (0.1%). The gel was washed for 30 min with Triton X-100 solution (2.5%) at 37 °C and incubated for 16 h in developing buffer (or digestion buffer; composition: 5 mM CaCl 2 , 0.02% Brij, and 50 mM Tris-HCl, pH 7.5) at 37 °C. The gel was stained for 30 min in 0.25% Coomassie Brilliant Blue (containing 40% methanol and 7% acetic acid). Areas of degradation were measured using an image analyzer (as clear bands against a darkly stained background) (Fuji-Film Corporation). Band densities were determined using multi-gauge image analysis software (Multi Gauge v.3.0; Fuji-Film Corporation).
RT-qPCR. RNA was isolated from cells using TRIzol reagent (RNAiso Plus; Takara Bio, Inc., Shiga, Japan) and extracted using a FastPure RNA Kit (Takara Bio, Inc.). cDNA was synthesized using a PrimeScript RT reagent Kit (Takara Bio, Inc.) with heating at 37 °C for 15 min and then 85 °C for 5 s. mRNA levels were analyzed by qPCR using Power SYBR Green PCR Master Mix and the ABI PRISM 7900 sequence detection system (Applied Biosystems; Thermo Fisher Scientific.). The PCR amplification primers used were as follows: MMP-9 (NM 004994) CCT GGA GAC CTG AGA ACC AATCT (sense) and CCA CCC GAG TGT AAC CAT AGC (antisense); SIRT6 (NM 001193285) CTT GGC ACA TTC TTC CAC AA (sense) and GCT TCC TGG TCA GCC AGA (antisense); GAPDH (NM 002046) ATG GAA ATC CCA TCA CCA TCTT (sense) and CGC CCC ACT TGA TTT TGG (antisense). PCR was conducted over 40 cycles at 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. The data were normalized to GAPDH to control for differences in target mRNA concentration. Quantitation was conducted using the comparative Ct method 18 . Luciferase assay. Cells were seeded into 24-well plates and transfected with AP-1 or NF-κB reporter plasmids (provided by Professor Kim Chul Ho, SungKyunKwan University, Suwon, Korea) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Transfected cells were treated with TPA or TNF-α for 3 h. Luciferase reporter assays were conducted using the dual luciferase assay kit (Promega Corporation) according to the manufacturer's instructions, and fluorescence intensities were measured using a luminometer (Lumat LB 9507, EG & G Berthold, Gaithersburg, MD, USA).
Invasion assay. Invasion assays were conducted using 24-well chambers (8 μm pore size) in which the upper side of the Transwell insert was coated at 37 °C for 30 min with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Cells were placed in the upper chambers, and the lower chambers were filled with conditioned medium containing TPA or TNF-α. After incubation for 24 h, the cells in the upper chambers were cleared using cotton swabs. The invaded cells on the bottom of the filter were fixed with 3.7-4.0% formalin for 10 min at room temperature and stained with crystal violet for 40 min at room temperature. Invading cells were counted in five randomly selected fields under a light microscope at 40 × magnification.
Migration assay. Migration assays were also conducted using 24-well chambers (8 μm pore size). Migration assays were performed in chambers without Matrigel. Cells were delivered to the upper chambers, and the lower chambers were filled with conditioned medium containing TPA or TNF-α. After incubation for 24 h, cells in the upper chambers were cleared using cotton swabs. The migrated cells on the bottom of the filter were fixed with 3.7%-4.0% formalin for 10 min at room temperature and stained with crystal violet for 40 min at room temperature. Moving cells were counted in five random fields under a light microscope at 40 × magnification.
Statistical analysis. Data

Results
MMP-9 and SIRT6 expression in human breast cancer tissue was higher than normal tissue. To assess MMP-9 and SIRT6 expression in clinical patients, protein levels in breast cancer tissues were analyzed by western blotting. The results showed that SIRT6 and MMP-9 expression in all malignant tissues was significantly higher than that in paired normal breast tissues (Student's t-test, p < 0.005; Fig. 1a,b).
To investigate the molecular mechanisms responsible for TPA-or TNF-α-induced activation of MMP-9, MCF-7 and MDA-MB-231 cells were pretreated with pharmacological inhibitors of MAPK signaling pathways. The MAPK inhibitors used were PD98059 (ERK inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor). TPA-or TNF-α-induced MMP-9 secretion and protein expression were reduced by each 20 μM concentration of three MAPK inhibitors in both cell lines ( Supplementary Fig. 2a,b). We investigated the effects of SIRT6 on the MAPK signaling pathway in MCF-7 and MDA-MB-231 cells. Additionally, TPA significantly increased the phosphorylation of ERK, JNK, and p38 in both cell lines, and SIRT6 knockdown decreased the phosphorylation of ERK and JNK in MCF-7 cells, and inhibited the phosphorylation of ERK, JNK, and p38 in MDA-MB-231 cells; however, total protein levels remained unaltered (Fig. 5a). TNF-α increased the phosphorylation of ERK, JNK, and p38 in both cell lines, and SIRT6 knockdown decreased the phosphorylation of ERK, JNK, and p38 in MCF-7 cells, and reduced the phosphorylation of ERK and p38 in MDA-MB-231 cells; however, total protein levels remained unaltered (Fig. 5b). These results showed that silencing of SIRT6 reduced phosphorylation of MAPK and reduction of MAPK activity correlated with reduced MMP-9 expression. SIRT6 knockdown suppressed TPA-or TNF-α-induced MMP-9 expression by reducing the activation of NF-κB and AP-1. To further understand the mechanisms responsible for MMP-9 transcriptional regulation, MCF-7 and MDA-MB-231 cells were pretreated with inhibitors of NF-κB (Bay 11-7092) or AP-1 (SR 11302) to examine the effects of MMP-9 on the activation of NF-κB and AP-1. Inhibition of NF-κB or AP-1 blocked TPA-or TNF-α-induced increases in MMP-9 secretion and protein levels ( Supplementary Fig. 2c,d).
In addition, the effects of SIRT6 on the activation of NF-κB and AP-1 were investigated in MCF-7 and MDA-MB-231 cells. Nuclear translocation of p65 and p50 (subunits of NF-κB) and phosphorylation of c-Jun and c-Fos (subunits of AP-1), as well as cytoplasmic phosphorylation of IKKα/β and degradation of IκBα (a subunit of NF-κB), were increased by TPA treatment. SIRT6 knockdown reduced the nuclear translocation of p65, p50, p-c-Jun, and p-c-Fos and reduced the cytoplasmic levels of p-IKKα/β and degradation of IκBα. Total c-Fos and c-Jun levels in the nucleus and total IKKα and IKKβ levels in the cytosol did not exhibit any changes (Fig. 6a). SIRT6 knockdown suppressed the TNF-α-induced nuclear translocation of p65, p50, p-c-Jun, and p-c-Fos, and inhibited the phosphorylation of cytoplasmic IKKα/β and degradation of IκBα. Total c-Fos and c-Jun in the nucleus and total IKKα and IKKβ in the cytosol did not exhibit any changes (Fig. 6b). The MMP-9 promoter consists of AP-1-and NF-κB binding sites; these transcription factors participate in the activation of the MMP-9 gene by TPA or TNF-α treatment. Luciferase assays for evaluation of the transactivation activities of NF-κB and AP-1 after TPA or TNF-α treatment showed that these interactions were significantly reduced in SIRT6knockdown cells (Fig. 6c,d). These results suggested that SIRT6 knockdown suppressed MMP-9 expression by reducing the activation of NF-κB and AP-1.  . (a, b) The amount of MMP-9 secreted into the medium was analyzed by gelatin zymography (zymo-MMP-9). MMP-9 levels were analyzed by western blotting using β-actin as an internal control. (c, d) MMP-9 mRNA levels were examined by RT-qPCR, using GAPDH as an internal control. The results are presented as means ± standard error of three independent experiments. *p < 0.05, vs. untreated controls. RT-qPCR reverse transcription quantitative polymerase chain reaction.

SIRT6 knockdown suppressed TPA-or TNF-α-induced Matrigel invasion and chamber migration.
We investigated the effects of SIRT6 on the capability of MCF-7 and MDA-MB-231 cells to degrade matrigel using Matrigel Transwell assays. The results showed that TPA and TNF-α significantly increased cell invasion, and SIRT6 knockdown significantly reduced the TPA-or TNF-α-induced increase in cell invasion in both cell lines (Fig. 7a,b). A migration assay was performed in a chamber without Matrigel. While determining the involvement of SIRT6 in breast cancer cell migration, SIRT6 knockdown was found to reduce TPA-or TNFα-induced cell migration in both cell lines (Fig. 7c,d). These results showed that SIRT6 suppressed the malignant invasion and migration potential of MCF-7 and MDA-MB-231 cells.

Discussion
Breast cancer is the most common cancer in the world and the leading cause of cancer death in female. Regulation of metastasis in breast cancer has been a major goal for successful treatment because most breast cancer-related deaths are due to advanced disease and progressive metastasis 19 . Cancer cell metastasis is a multi-step process that requires tumor cell invasion, migration to the circulation, extravasation, and growth in the metastatic region 20 . Invasion and migration are considered to be the most critical factors in primary tumor metastasis 3 . Here, we obtained the novel findings that SIRT6 is involved in the modulation of breast cancer cell invasion and migration by regulating MMP-9 expression in breast cancer cells, suggesting that SIRT6 is a novel target molecule for the prevention of breast cancer. Sirtuins play key roles in various biological processes, including tumor progression. Seven sirtuins (SIRT1 to SIRT7) have been identified 21 , and SIRT6 has been reported to participate in tumor suppression and promotion. Tumor promotion by SIRT6 has been reported in several types of malignancies 22 ; however, further studies are required to determine the role of SIRT6 in cancer invasiveness. In breast cancer cells, SIRT6 has been related to the upregulation of MMP-9 20 , and SIRT6 knockdown has been shown to reduce MMP-9 expression 21 . In our   www.nature.com/scientificreports/ study, SIRT6 additively upregulated TPA-and TNF-α-induced MMP-9 expression and silencing of SIRT6 reduced TPA-and TNF-α-induced MMP-9 expression (Figs. 3 and 4). These data showed that SIRT6 involved in MMP-9 expression. MMP-9 is a major member of the zinc metalloproteinase family because it stimulates cancer metastasis by degrading ECM 23 and collagens, facilitating cancer cell invasion and metastasis 24 . Therefore, MMP-9 is a potentially key molecule in cancer invasion 25 and is considered a target for drug development 26 . The results obtained in the present study revealed that SIRT6 is involved in the modulation of breast cancer cell invasion and migration by regulating MMP-9 expression in MCF-7 and MDA-MB-231 cells.
The synthesis and secretion of MMP-9 are strictly regulated by various biological factors 27 . Several studies have reported that TPA activates the synthesis and secretion of MMP-9 in breast cancer cells 28 . TPA induces inflammatory responses and acts as a tumor promoter that stimulates PKC isozymes by direct binding. TPA dramatically induces the invasion of human breast cancer cells by upregulating MMP-9 expression via transcription factors and MAPK pathways 29 . In addition, TNF-α is known to increase tumor cell migration and invasion, and is involved in all steps of tumorigenesis 30 . TNF-α-induced tumor initiation and tumor promotion are mediated by MMP-9-induced activation of NF-κB and AP-1-dependent signals in tumor cells 31 . Our study showed that TPA and TNF-α significantly upregulated MMP-9 expression in MCF-7 and MDA-MB-231 cells (Fig. 2). SIRT6 regulate the fatty acylation level of TNF-α and then the synthesis of TNF-α 32 . This study also suggested that SIRT6 upregulated TPA and TNF-α expression in MCF-7 and MDA-MB-231 cells (Figs. 3 and 4).
PKC activity is important for cancer cell migration and is correlated with MMP-9 expression in breast cancer cells. Our data showed that TPA or TNF-α mediated PKC activity, but we identified no associations involving PKC isoforms and SIRT6 in breast cancer cells (Supplementary Fig. 1). Thus, we focused on other mechanisms related to cancer progression and metastasis.
MMP-9 promoters have various transcription factor-binding sites, and AP-1 and NF-κB are key transcription factors 8 . Nuclear factors binding to the AP-1 and NF-κB promoters are induced and synergistically contributed by TPA and TNF-α in tumor cell invasiveness 33 . TNF-α activates the receptor tyrosine kinase pathway, leads to activation of NF-κB. And the AP-1 transcription factor is known to be key for MMP-9 expression. In addition, the downregulated p65 blocked TNF-α-induced MMP-9 expression and increase of IκBα blocked NF-κB activity and MMP-9 expression. This demonstrates that NF-κB activity is essential for upregulating MMP-9 expression 34 . Inhibition of MMP-9 and cell invasion involves inhibition of the MAPK pathway 35 . SIRT6 is known to regulate the activation of MAPKs signaling-correlated genes by deacetylation. However, the functional role of SIRT6 deacetylase activity in the MAPK signaling pathway is yet to be fully understood. SIRT6 positively modulated the levels of phosphorylated ERK and regulated MMP9 abundance probably through the MAPK signaling pathway 36 . Our data also showed that silencing of SIRT6 modulated activation of MAPK expression (p-ERK, p-JNK, p-p38). These results indicated that SIRT6 involved MAPK signaling pathway. In order to better understand the signaling cascades underlying MMP-9 expression and SIRT6 modulation in MCF-7 and MDA-MB-231 cells, the effects of specific inhibitors of MAPKs, NF-κB, and AP-1 were assessed. MMP-9 expression were significantly inhibited by MAPKs, NF-κB, and AP-1 ( Supplementary Fig. 2). In addition, the transactivation activities of NF-κB and AP-1 were reduced, and MAPK phosphorylation was suppressed by SIRT6 knockdown; therefore, MMP-9 expression was downregulated. These results showed that MMP-9 is mainly regulated by the MAPK, NF-κB, and AP-1 signaling pathways, although it should be noted that not all MAPKs are involved (Figs. 5 and 6). The anti-invasive effect of SIRT6 can be attributed to the suppression of MAPK signaling, NF-κB and AP-1 activation, and MMP-9 expression.
In conclusion, SIRT6 regulated the migration and invasion of breast cancer cells in vitro and played an essential role in TPA-or TNF-α-induced MMP-9 expression. Furthermore, SIRT6 knockdown suppressed TPA-or TNF-α-induced MMP-9 expression by inhibiting the MAPK, AP-1, and NF-κB signaling pathways in MCF-7 and MDA-MB-231 cells. This study suggests that SIRT6 is a novel target molecule for the prevention of breast cancer invasion and metastasis.  . (a, b) MCF-7 and MDA-MB-231 cells were transfected with control siRNA or SIRT6 siRNA and then treated with TPA or TNF-α for 3 h. Cytosolic and nuclear extracts were prepared and nuclear protein extracts were analyzed by western blotting using antibodies against p65, p50, p-c-Fos, p-c-Jun, c-Fos, and c-Jun, and cytosolic protein extracts were examined by western blotting using antibodies against IκBα, p-IκBα, IKKα, IKKβ, and p-IKKα/β. PCNA was used as a nuclear loading control, and β-actin was used as an internal control for cytoplasmic protein detection. (c, d) NF-κB-luc and AP-1-luc reporters were co-transfected with luciferase thymidine kinase (Renilla) reporter into both cell lines. Thereafter, cells were transfected with control siRNA or SIRT6 siRNA and treated with TPA or TNF-α for 4 h. NF-κB and AP-1 promoter activities were examined using a dual-luciferase reporter assay. The results are presented as means ± standard error of three independent experiments. *p < 0.05, vs. TPA-or TNF-α-treated control siRNA. Renilla Renilla luciferase thymidine kinase reporter. The migration chamber was then implemented without Matrigel. Cells were seeded into the upper chambers, and TPA or TNF-α was added to the lower chambers. After treatment for 24 h, the cells were photographed under a light microscope at 40 × magnification. The results are shown as the mean number of migrated cells, and were obtained by counting migrated cells in five randomly selected fields. The results are presented as means ± standard error of three independent experiments. **p < 0.01 vs. TPA-or TNF-α-treated control siRNA.